肠道病毒71型及甲乙型肝炎联合疫苗免疫原性的初步评价

发布时间：2018-03-04 21:09

本文选题：肠道病毒71型　切入点：甲型肝炎病毒　出处：《北京协和医学院》2015年硕士论文　论文类型：学位论文

【摘要】：目的初步评价肠道病毒71型(Enterovirus 71, EV71)、甲型肝炎(甲肝)病毒(Hepatitis A, HAV)与乙型肝炎(乙肝)病毒(Hepatitis B, HBV)联合疫苗中三种抗原之间的相互作用及其免疫小鼠诱导的特异性免疫应答水平。方法将EV71灭活疫苗抗原、HAV灭活疫苗抗原及重组HBV疫苗抗原(HBsAg)按常规剂量,以直接吸附法吸附AI(OH)3佐剂,配制成EV71-HAV-HBV联合疫苗,并设二联疫苗组、单苗组、佐剂对照组和空百对照组(PBS)。分别于0、28d各经腓肠肌免疫BALB/c小鼠一次,于初免后2、4周和第二剂免疫后2、4、8、16、28周经尾动脉采血,分离血清。采用微量中和试验检测血清EV71中和抗体效价；采用酶联免疫吸附实验(enzyme-linked immunosorbent assay, ELISA)检测血清中Anti-HBs、Anti-HAV水平。于初免后3、4周和第二剂免疫后第1、2、4周经尾动脉采血,用流式细胞仪检测小鼠全血中淋巴细胞亚群CD4+/CD8+的百分率及小鼠血清中TNF、IFNy、IL-5、IL-4、IL-2含量,同时取小鼠脾脏,分离得到脾单个核细胞(mononuclear cell, MNC),采用酶联免疫斑点试验(enzyme-linked immunospot assay, ELISPOT)检测MNC中抗原特异性FNy的分泌水平。结果各疫苗组小鼠血清Anti-HBs和Anti-HAV阳转率分别在第二剂免疫后第2周和第4周达100%,EV71中和抗体只有部分组达到100%；佐剂对照组和空白对照组抗体均无阳转。初免后第4周,疫苗组CD3+、CD4+细胞百分率较高,第5周均有不同程度的下降；CD8+细胞百分率第3周最高,第4周有不同程度的下降。第二剂免疫后第1周,三联疫苗组5种细胞因子水平较其他组高,提示三联疫苗组在第二次抗原刺激时能较早的产生细胞因子相关的免疫应答。各疫苗组分泌IFNy的斑点形成细胞(spot forming cell, SFC)数在5个检测时点有所波动,第二剂免疫后第1周,HBsAg多肽和EV71全病毒刺激产生的IFNγ分泌阳转率均达到100%,同时SFC数也达到峰值；HAV全病毒刺激产生的IFN-γ水平在5个时间点的检测结果均为阴性；佐剂和空白对照组的ELISPOT检测结果均为阴性。结论一定剂量的EV71-HAV-HBV联合疫苗具有较好的免疫原性,可迅速诱导小鼠产生良好的体液免疫应答和一定水平的细胞免疫应答,三种抗原间未发现相互作用。
[Abstract]:Objective to evaluate the interaction between enterovirus 71, EV71, hepatitis A virus hepatitis A (HAV) and hepatitis B (HBV) vaccine and the specificity of immunizing mice. Methods EV71 inactivated vaccine antigen and recombinant HBV vaccine antigen were used in routine dose. A combined EV71-HAV-HBV vaccine was prepared by adsorption of AI(OH)3 adjuvant by direct adsorption. The mice were divided into two groups: single vaccine group, single vaccine group, adjuvant control group and blank 100 control group. BALB/c mice were immunized with gastrocnemius muscle on day 0 and 28, respectively. Blood samples were collected from caudal artery at the 4th week after the first immunization and 2 weeks after the second dose immunization. The neutralizing antibody titers of serum EV71 were detected by microneutralization test. Enzyme linked immunosorbent assay (Elisa) was used to detect the level of Anti-HBsAg Anti-HAV in serum. Blood samples were collected from caudal artery at 3 ~ 4 weeks after immunization and 1 ~ 2 ~ 4 weeks after the second dose immunization. Flow cytometry was used to detect the percentage of CD4 / CD8 in the whole blood of mice and the level of IL-2 in serum of mice. The spleen of the mice was taken at the same time. Splenic mononuclear cells (MNCs) were isolated and detected by enzyme-linked immunospot assay (ElisPOT) for the secretion of antigen-specific FNy in MNC. Results the serum Anti-HBs and Anti-HAV positive conversion rates of mice in each vaccine group were determined in the second dose after the second dose immunization. At week 1 and week 4, the neutralizing antibody against EV71 was only partially divided into 100 parts, while the antibody in the adjuvant control group and the blank control group had no positive change. At the fourth week after the first immunization, no positive changes were found in the adjuvant control group and blank control group. In the vaccine group, the percentage of CD4 cells of CD3 was higher, the percentage of CD8 cells decreased in different degrees at the 5th week, the percentage of CD8 cells decreased at the 3rd week and decreased at the 4th week, and decreased at the first week after the second dose immunization. The levels of 5 cytokines in the triple vaccine group were higher than those in the other groups. The results indicated that the triplex vaccine group could produce cytokine related immune response earlier during the second antigen stimulation. The number of spot forming cells that secreted IFNy in each vaccine group fluctuated at 5 test time points. In the first week after the second dose of immunization, the positive conversion rate of IFN 纬 was 100, and the number of SFC reached the peak value. The level of IFN- 纬 produced by the whole virus stimulation was negative at 5 time points. The results of ELISPOT detection in adjuvant and blank control group were negative. Conclusion certain dose of EV71-HAV-HBV combined vaccine has good immunogenicity and can induce a good humoral immune response and a certain level of cellular immune response in mice. No interaction was found among the three antigens.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R392-33

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